RESUMO
BACKGROUND: Enumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system. METHODS: The HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs). RESULTS: Using genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs. CONCLUSION: HyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood.
Assuntos
Neoplasias da Mama , Células Neoplásicas Circulantes , Humanos , Feminino , Células Neoplásicas Circulantes/patologia , Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , RNA Mensageiro/metabolismo , RNA , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismoRESUMO
OBJECTIVE: The purpose of this study was to establish normal ranges for human epididymis protein 4 (HE4) serum levels in healthy women. STUDY DESIGN: HE4 levels were measured in healthy women and analyzed by age, menopausal status, and pregnancy status. Upper 95th percentiles were determined for normal ranges. RESULTS: Serum samples from 1101 healthy women and 67 pregnant women were analyzed. Above the age of 40 years significant elevations in HE4 concentrations emerged with advancing age. The upper 95th percentile for HE4 levels was 89 pmol/L for premenopausal women, 128 pmol/L for postmenopausal women, and 115 pmol/L for all women. There was a significant difference in the median serum HE4 levels in premenopausal women (46.6 pmol/L) compared with postmenopausal women (57.6 pmol/L; P < .001). In pregnant women, median HE4 concentrations were significantly lower than their premenopausal counterparts (P < .001). CONCLUSION: HE4 serum concentrations vary significantly on the basis of age. These variations must be considered when the upper limit of normal for HE4 is determined.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma/sangue , Neoplasias Ovarianas/sangue , Proteínas/análise , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-Idade , Pós-Menopausa/sangue , Gravidez , Pré-Menopausa/sangue , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
OBJECTIVE: The human epididymis protein 4 (HE4) is a novel biomarker for ovarian cancer. This study measured the HE4 and CA125 levels in women with benign gynecological disorders. STUDY DESIGN: Sera were obtained from women prior to surgery for a pelvic mass and HE4 and CA125 levels were determined. The proportions of patients with elevated biomarker levels were compared. RESULTS: There were 1042 women with benign disease. HE4 levels were less often elevated than CA125 (8% vs 29%, P < .001). A marked difference was observed in patients with endometriosis in which HE4 was elevated in 3% of patients and CA125 in 67% (P < .0001). Serous ovarian tumors were associated with elevated levels of HE4 in 8% of patients and CA125 in 20% (P = .0002); uterine fibroids in 8% vs 26% (P = .0083); dermoids in 1% vs 21% (P = .0004); and inflammatory disease in 10% vs 37% (P = .014). CONCLUSION: HE4 is elevated less frequently than CA125 in benign disease, particularly in premenopausal patients.
Assuntos
Antígeno Ca-125/sangue , Doenças dos Genitais Femininos/sangue , Proteínas de Membrana/sangue , Proteínas/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
OBJECTIVE: To evaluate the detection of malignancy in women with a pelvic mass by using multiplexed gene expression analysis of cells captured from peripheral blood. METHODS: This was an IRB-approved, prospective clinical study. Eligible patients had a pelvic mass and were scheduled for surgery or biopsy. Rare cells were captured from peripheral blood obtained preoperatively by using a microfluidic cell capture device. Isolated mRNA from the captured cells was analyzed for expression of 72 different gene transcripts. Serum levels for several commonly assayed biomarkers were measured. All patients had a tissue diagnosis. Univariate and multivariate logistic regression analyses for the prediction of malignancy using gene expression and serum biomarker levels were performed, and receiver operating characteristic curves were constructed and compared. RESULTS: A total of 183 evaluable patients were enrolled (average age 56 years, range 19-91 years). There were 104 benign tumors, 17 low malignant potential tumors, and 62 malignant tumors. Comparison of the area under the receiver operating characteristic curve for individual genes and various combinations of genes with or without serum biomarkers to differentiate between benign conditions (excluding low malignant potential tumors) and malignant tumors showed that a multivariate model combining the expression levels of eight genes and four serum biomarkers achieved the highest area under the curve (AUC) (95.1%, 95% CI 92.0-98.2%). The MAGIC (Malignancy Assessment using Gene Identification in Captured Cells) algorithm significantly outperformed all individual genes (AUC 50.2-65.2%; all P <.001) and a multivariate model combining 14 different genes (AUC 88.0%, 95% CI 82.9-93.0%; P =.005). Further, the MAGIC algorithm achieved an AUC of 89.5% (95% CI 81.3-97.8%) for stage I-II and 98.9% (95% CI 96.7-100%) for stage III-IV patients with epithelial ovarian cancer. CONCLUSION: Multiplexed gene expression evaluation of cells captured from blood, with or without serum biomarker levels, accurately detects malignancy in women with a pelvic mass. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT02781272. FUNDING SOURCE: This study was funded by ANGLE Europe Limited (Surrey Research Park, Guildford, Surrey, United Kingdom).
Assuntos
Antígeno Ca-125 , Neoplasias Ovarianas , Humanos , Feminino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Estudos Prospectivos , Biomarcadores Tumorais , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , AlgoritmosRESUMO
Abnormal DNA content in tumor cells represents large scale chromosomal alterations and reflects later changes of genetic instability. Her-2/neu oncogene is amplified in 20-30% of breast and ovarian cancer patients and is associated with poor prognosis. Therefore, we evaluated prognostic value of Her-2/neu expression and DNA content measurements in 252 clinically localized PCa patients with long-term follow-up after radical prostatectomy for progression, metastasis and PCa-specific death. Her-2/neu expression was determined by immunohistochemistry and DNA content measurements employed Feulgen-stained cancer nuclei captured using static image cytometry system. Cox proportional hazard regression and Kaplan-Meir plots were used to identify significant prognostic factors for progression, metastasis and PCa-specific death. The proportions of Her-2/neu positive and high %DNA index tumors significantly increased from nonprogressor to progressors without metastasis to progressors with metastasis (p < 0.0001; <0.0001). Further, the proportions of Her-2/neu positive and high %DNA index tumors significantly increased from patients who died from another cause without progression to those who died from another cause with progression to those died with PCa-specific death (p = 0.027; <0.0001). Her-2/neu expression and %DNA index were significant prognosticators for progression (p Assuntos
DNA/genética
, Regulação Neoplásica da Expressão Gênica/genética
, Prostatectomia
, Neoplasias da Próstata/metabolismo
, Neoplasias da Próstata/patologia
, Receptor ErbB-2/metabolismo
, Progressão da Doença
, Seguimentos
, Humanos
, Masculino
, Pessoa de Meia-Idade
, Metástase Neoplásica/patologia
, Prognóstico
, Neoplasias da Próstata/genética
, Neoplasias da Próstata/cirurgia
, Receptor ErbB-2/genética
, Taxa de Sobrevida
, Fatores de Tempo